The requirement for molecular chaperones during endoplasmic reticulum-associated protein degradation demonstrates that protein export and import are mechanistically distinct
The requirement for molecular chaperones during endoplasmic reticulum-associated protein degradation demonstrates that protein export and import are mechanistically distinct
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DOI：
10.1074/jbc.274.6.3453
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发表时间：
1999-02-05
影响因子：
4.8
通讯作者：
McCracken, AA
中科院分区：
文献类型：
--
作者：
Brodsky, JL;Werner, ED;McCracken, AA
多肽进入酵母内质网（ER）需要两个hsp70，Ssa1p在胞质溶胶和BiP（Kar2p）在ER腔。输入后，异常多肽可被输出到细胞质中，由蛋白酶体降解，ER伴侣钙连接蛋白（Cne1p）的缺陷会损害其降解。进口和出口都需要BiP和Sec61p易位复合物，这表明进口和出口可能是机械相关的。我们现在表明，cne1三角洲和两个kar2突变体等位基因表现出合成的相互作用，并在kar2突变体微粒体的出口和前α因子的降解是有缺陷的。脉冲追踪分析表明，A1PiZ，另一种底物的降解，是稳定的kar2菌株在限制性温度。由于两个kar2突变体检查是精通多肽进口，在ER蛋白的出口和进口的BiP的作用不同，这表明这些过程必须是机械上不同的。为了检查Ssa1p是否从ER驱动多肽并且也是降解所需的，我们使用在SSA1中含有突变或其中Ssa1p的水平可以被调节的菌株组装反应。我们发现，前α因子和A1PiZ降解正常，进一步表明，进口和出口是不同的，其他胞质因子可能拉多肽从ER。
Polypeptide import into the yeast endoplasmic reticulum (ER) requires two hsp70s, Ssa1p in the cytosol and BiP (Kar2p) in the ER lumen. After import, aberrant polypeptides may be exported to the cytoplasm for degradation by the proteasome, and defects in the ER chaperone calnexin (Cne1p) compromise their degradation. Both import and export require BiP and the Sec61p translocation complex, suggesting that import and export may be mechanistically related. We now show that the cne1 Delta and two kar2 mutant alleles exhibit a synthetic interaction and that the export and degradation of pro-alpha factor is defective in kar2 mutant microsomes. Pulse-chase analysis indicates that A1PiZ, another substrate for degradation, is stabilized in the kar2 strains at the restrictive temperature. Because two of the kar2 mutants examined are proficient for polypeptide import, the roles of BiP during ER protein export and import differ, indicating that these processes must be mechanistically distinct. To examine whether Ssa1p drives polypeptides from the ER and is also required for degradation, we assembled reactions using strains either containing a mutation in SSA1 or in which the level of Ssa1p could be regulated. We found that pro-alpha factor and A1PiZ were degraded normally, indicating further that import and export are distinct and that other cytosolic factors may pull polypeptides from the ER.